Arabidopsis‐expressing lysine‐null SUMO1 reveals a non‐essential role for secondary SUMO modifications in plants

Abstract The reversible conjugation of small ubiquitin‐like modifier (SUMO) to other proteins has pervasive roles in various aspects of plant development and stress defense through its selective attachment to numerous intracellular substrates. An intriguing aspect of SUMO is that it can be further modified by SUMOylation and ubiquitylation, which isopeptide‐link either or both polypeptides to internal lysines within previously bound SUMOs. Although detectable by mass spectrometry, the functions of these secondary modifications remain obscure. Here, we generated transgenic Arabidopsis that replaced the two related and essential SUMO isoforms (SUMO1 and SUMO2) with a lysine‐null SUMO1 variant (K0) immune to further SUMOylation/ubiquitylation at these residues. Remarkably, homozygous SUMO1(K0) sumo1 sumo2 plants developed normally, were not hypersensitive to heat stress, and have nearly unaltered SUMOylation profiles during heat shock. However, subtle changes in tolerance to salt, paraquat, and the DNA‐damaging agents bleomycin and methane methylsulfonate were evident, as were increased sensitivities to ABA and the gibberellic acid biosynthesis inhibitor paclobutrazol, suggesting roles for these secondary modifications in stress defense, DNA repair, and hormone signaling. We also generated viable sumo1 sumo2 lines expressing a SUMO1(K0) variant specifically designed to help isolate SUMO conjugates and map SUMOylation sites, thus offering a new tool for investigating SUMO in planta.

with other proteins, and/or stimulating or blocking subsequent modification by Ub. In many cases, the appended SUMO regulates proteinprotein interactions through its binding to other proteins harboring SUMO-interacting Motifs (SIMs) (Yau et al., 2021). Ultimately, numerous cellular events are impacted, many of which are important to genome organization, transcription, RNA splicing and metabolism, signal transduction, and plant stress defense Benlloch & Lois, 2018;Morrell & Sadanandom, 2019).
SUMO addition occurs through an ATP-dependent conjugation cascade sequentially involving the heterodimeric SAE1/SAE2 SUMOactivating enzyme (E1), the SCE1 SUMO-conjugating enzyme (E3), and a small set of SUMO-ligases (E3s) that connect the charged, thiolester-linked E2-SUMO adduct with specific substrates and then facilitate SUMO transfer onto accessible lysines (Geiss-Friedlander & Melchior, 2007;Psakhye & Jentsch, 2012). The final product is a SUMO moiety covalently attached through an isopeptide bond between the C-terminal glycine carboxyl group of SUMO and one or more ε-amino lysl groups within the substrate. Arabidopsis encodes at least three E3 types-SIZ1, MMS21, and PIAL1 and 2, which ultimately help modify more than a thousand substrates as detected by mass spectrometry (MS) (Miller et al., 2010(Miller et al., , 2013Rytz et al., 2018).
In some cases, SUMOylation can also occur on multiple proteins in close proximity to the substrate directly recognized by the E3 through a SUMO "spray" mechanism (Psakhye & Jentsch, 2012). The pathway also includes a family of deSUMOylating enzymes that can release both the substrate and SUMO moieties in free, functional forms by specific cleavage of the isopeptide bond, thus allowing for reversible modifications (Kunz et al., 2018;Morrell & Sadanandom, 2019).
Plants typically express several SUMO isoforms. Within the eightmembered family in Arabidopsis, only four appear to be transcriptionally active (Kurepa et al., 2003;van den Burg et al., 2010). Included are the close paralogs-SUMO1 and SUMO2 (94% identical), which are best expressed, essential genetically, and responsible for most, if not all, of the SUMOylation seen in vivo especially under stress such as heat shock (Augustine et al., 2016;Kurepa et al., 2003;Saracco et al., 2007). SUMO3 and SUMO5 by contrast, are loosely constrained by sequence (only 48% and 35% identical to SUMO1) and expressed at low levels with little evidence of functionality besides a possible role for SUMO3 in salicylic acid-mediated defense and/or antagonizing the actions of SUMO1/2 (Ingole et al., 2021;van den Burg et al., 2010). It still remains to be conclusively demonstrated that SUMO3 and SUMO5 actually become conjugated to other proteins in planta.
Like Ub, an intriguing feature of the bound SUMO is that it can be further modified by SUMOylation and/or ubiquitylation in which the first SUMO becomes modified at accessible lysines by additional SUMOs or Ubs (Keiten-Schmitz et al., 2019;Vertegaal, 2010). Ultimately, it is possible that chains of SUMO or SUMO-Ub are assembled with either linear or branched architectures using these lysineglycine isopeptide connections. In yeast and mammals, polymer concatenation mostly occurs at a flexible 20-amino-acid N-terminal extension upstream of the β-grasp Ub-fold in SUMO, which is predicted to be intrinsically disordered ( Figure S1a). Assembly of SUMO-SUMO chains is likely encouraged by SIZ1-and PIAL1/2-type E3s that harbor binding sites for both the E2-SUMO adduct and for free SUMO via SIMs (Jansen & Vertegaal, 2021;Keiten-Schmitz et al., 2019). For SUMO-Ub linkages, a family of SUMO-targeted Ub ligases (StUbLs) directs Ub addition onto previously bound SUMOs, which includes human RNF4 and RNF11 and the yeast Slx5/8 heterodimer (Kumar et al., 2017). Assembly of the SUMO-SUMO polymers can be counterbalanced by a subfamily of deSUMOylating enzymes that includes Semp6 and Semp7 in humans and Ulp2 in yeast, thereby reversing this secondary modification (Kunz et al., 2018).
At present, the identities of the proteins modified with SUMO-SUMO and SUMO-Ub polymers are mostly unclear as are the functions of the added SUMOs/Ubs (Keiten-Schmitz et al., 2019;Vertegaal, 2010). Through the analysis of SUMO mutants that replaced all lysines with arginines and thus blocked subsequent additions, it appears that such secondary modifications are not essential in the yeast Saccharomyces cerevisiae, with the caveat that a deletion mutant of the Upl2 isopeptidase which cannot disassemble such polymers is phenotypically compromised and hyperaccumulates high-molecular mass SUMO conjugates (Bylebyl et al., 2003). In Schizosaccharomyces pombe, lysine-null SUMO strains are also viable but display nuclear defects and a hypersensitivity to genotoxic stress (Skilton et al., 2009). In mammalian cells, SUMO-Ub chains have been implicated in various aspects of centromere organization, chromatin cohesion, DNA repair, and replication likely through the action of StUbLs (Jansen & Vertegaal, 2021;Keiten-Schmitz et al., 2019). Whether such polymers are essential to mammals remains unclear but they preferentially assemble in cells under stress, suggesting protective functions (Vertegaal, 2010).
In plants, the actions of SUMO-SUMO and SUMO-Ub concatemers are not yet understood. Miller et al. (2010Miller et al. ( , 2013  326 m/z) or Ub (GG; 114 m/z) fragments. At least for the assembly of SUMO-Ub adducts, a small gene family encoding possible orthologs of yeast and mammalian StUbLs have been detected in plant genomes (six in Arabidopsis); one could partially rescue the genome integrity defects seen in the rfp1/rfp2 StUbL mutant from S. pombe (Benlloch & Lois, 2018;Elrouby, 2015;Elrouby et al., 2013), while another has been indirectly linked to histone methylation (Hale et al., 2016).
Here, we attempted to address the importance of SUMO-SUMO and SUMO-Ub polymers through the creation of Arabidopsis SUMO1/2 lines potentially missing these secondary modifications.
This was accomplished by rescuing mutants eliminating the pair with a SUMO1 variant harboring arginine replacements for all seven lysines, which would block these additions but still retain functionality at least as seen in vitro (Augustine et al., 2016;Zhang et al., 2021). Remarkably, these SUMO1(K0) sumo1-1 sumo2-1 plants were phenotypically normal when grown under optimal conditions and showed similar SUMOylation patterns before, during, and after heat shock. However, altered stress tolerance and hormone sensitivities were seen, suggesting that these secondary SUMO modifications do have more subtle roles. We also generated Arabidopsis lines expressing a 6His-tagged SUMO1(K0) mutant bearing substitutions designed to help map SUMO1/2 conjugation sites (Miller et al., 2010(Miller et al., , 2013. Preliminary studies revealed that this 6His-(M1R)-K0(H89R) variant also retained functionality in planta, thus providing a new tool to dissect SUMOylation by MS/MS approaches.

| SUMO lysines are not essential in Arabidopsis
The canonical SUMO1/2 isoforms in plants harbor seven strictly conserved lysines (K9, K10, K21, K23, K35, K41, and K42; Figure S1B) that are likely targets of secondary SUMOylation or ubiquitylation, with prior MS/MS analyses of the Arabidopsis SUMOylome confirming that K23 and K42 can be modified in planta (Miller et al., 2010(Miller et al., , 2013. Based on our prior studies showing that these lysines are not essential for reversible conjugation at least in vitro (Augustine et al., 2016;Zhang et al., 2021), we sought to understand their impact on secondary SUMO modifications by generating Arabidopsis SUMO1 variants either wild type for the sequence or harboring lysine ! arginine substitutions at K23 and K42 (K23,42-R) or at all seven lysines (K0) (Figure 1a). These transgenes, expressed under the control of the native SUMO1 promoter, were then introduced into a previously described null sumo1-1 and sumo2-1 background generated by T-DNA insertional mutagenesis (Miller et al., 2010;Saracco et al., 2007).
As homozygous sumo1-1 sumo2-1 plants are inviable by arresting growth early in embryogenesis, these transgenes were introduced by Agrobacterium-mediated gene transfer into plants heterozygous for the sumo1-1 allele and homozygous for the sumo2-1 allele, and then screened for plants homozygous for all three loci by genomic polymerase chain reaction (PCR) of progeny from a self-cross (Miller et al., 2010). As shown in Figure 1b,c, the wild-type (WT) SUMO1 and F I G U R E 1 Lysine-null SUMO1 variant rescues the Arabidopsis embryo-lethal sumo1 sumo2 mutant. (a) Diagram of the pSUMO1:SUMO1(K0) transgene expressing a lysine-null variant of Arabidopsis SUMO1 in which all seven lysines were replaced with arginines (K0) (see Figure S1). (top) Organization of the SUMO1(K0) transgene. (bottom) Organization of the endogenous SUMO1 and SUMO2 loci showing the positions of the T-DNA insertions (red triangles) that disrupt expression. The SUMO1 promoter is shown in blue. Untranslated and coding regions are in yellow and orange boxes, respectively. Introns are shown by the broken lines. Half arrows locate the positions of the primers used for genotyping and qRT-PCR (qRT) in panels (b) and (c), respectively. (b) Genotype confirmation of Col-0 and the SUMO1 transgenic lines by PCR. Genomic DNA was isolated from 27-day-old wild-type Arabidopsis Col-0, and independent sumo1-1 sumo2-1 transgenic seedlings expressing wild-type SUMO (WT), the SUMO1(K0) variant (K0 #2, K0 #109, and K0 #116), or the K23,42-R variant harboring lysine!arginine substitutions at positions 23 and 42. (c) Relative transcript abundance for the SUMO1 loci in the genotypes described in panel (b). Transcript levels were quantified by qRT-PCR of total RNA extracted from 7-day-old seedlings, using the qRT primer pairs shown in panel (a) and those for the ACT2 mRNA as the internal standard. All values were normalized to that obtained with Col-0; bars represent the mean (±SD) from three technical replicates. Letters above the bars cluster significant differences by one-way ANOVA (p value <.05). (d) Growth on soil under a LD photoperiod of representative wild-type (Col-0), and transgenic lines expressing wild-type SUMO1 (WT) or the SUMO1(K0) and K23,42-R variants in the sumo1-1 sumo2-1 background. (top) plants at flowering 40 days after sowing. (bottom) Rosettes at 20 days after sowing. Growth of the amiS2 sumo1-1 line that suppressed SUMO2 expression by RNAi and eliminated SUMO1 expression by the sumo1-1 mutation (van den Burg et al., 2010) was included for comparison. Scale bars = 2 cm. the K23,42-R and K0 mutant transgenes, and the sumo1-1 and sumo2-1 mutations were readily detected by genomic PCR in T2 plants generated from such self-crosses. We subsequently confirmed presence of the transgenes by sequencing fragments amplified from genomic DNA using SUMO1-specific primers (Figure 1a,b); this sequencing detected either the WT sequence or the respective lysine ! arginine codon replacements. Quantitative reversetranscribed (q -sRT) PCR then identified a collection of sumo1-1 sumo2-1 lines that expressed the WT SUMO1 transgene or those encoding the K23,42-R or K0 variants at levels near equal to or exceeding those from the endogenous SUMO1/2 loci found in the Col-0 WT background (Figure 1c).
Rescues of the lethal sumo1-1 sumo2-1 combination by the K23,42-R and K0 variants were immediately obvious as triple homozygous plants could be readily identified as viable and fecund as opposed to homozygous sumo1-1 sumo2-1 embryos that abort soon after fertilization (Saracco et al., 2007). When grown under a long-day (LD)-photoperiod, 20-day-old rosettes expressing the mutant SUMOs were nearly indistinguishable from Col-0 rosettes and sumo1-1 sumo2-1 rosettes rescued with the WT SUMO1 transgene (Figure 1d).
Even for 40-day-old flowering plants, the K23,42-R-and K0-rescued lines were strikingly similar to Col-0 and those expressing WT SUMO1, except for a slight but reproducible reduction in inflorescence height without compromising the viability of the resulting seeds. This relatively normal growth was in contrast to an ami-S2 sumo1-1 line first described by van den Burg et al. (2010) that is null for SUMO1 but downregulated for SUMO2 expression by antisense RNAi co-suppression; it displayed stunted rosettes and dwarfed inflorescences when grown alongside the rescued mutants ( Figure 1d).

| Lysine-null SUMO1 generates SUMO conjugates in planta
One remarkable feature of SUMO1/2 is their robust conjugation to other proteins upon exposure of plants to acute heat stress (Augustine et al., 2016;Kurepa et al., 2003;Saracco et al., 2007).
Using this response as an assay for protein functionality, we compared by immunoblot assays the SUMOylation profiles of the plants expressing the K23,42-R and K0 variants to those expressing WT SUMO1 before and after a 37 C heat shock. As both free SUMO1/2 and their conjugates are typically detected with anti-SUMO1 antibodies after sodium dodecylsulphate-polyacrylamide-gel electrophoresis (SDS-PAGE) (Kurepa et al., 2003;Saracco et al., 2007), we considered Subsequent immunoblot analyses of total extracts prepared from seedlings grown at 24 C, and then exposed to a 30-min heat shock at 37 C followed by a return to 24 C, revealed only modest differences in the heat-shock-induced SUMOylome profile in sumo1-1 sumo2-1 seedlings expressing the K23,42-R and K0 variants as compared with Col-0 seedlings or those expressing WT SUMO1 (Figure 2c). Even the rise and fall of conjugates with molecular mass above 50 kDa appeared mostly coincident, with low levels of conjugates evident before the heat stress (obvious in more exposed immunoblots), high levels seen after the 30 min heat shock and immediately after an ensuing re-acclimation back to 24 C, and then a return back to low levels of conjugates after continued incubation at 24 C. In fact, all of the increases in conjugate levels were lost within 3.5 h at the cooler temperature.
An interesting feature of heat-induced SUMOylation is that the levels of conjugates are directly proportional to the amount of free SUMO, indicating that the response is SUMO1/2 limiting (Kurepa et al., 2003;Saracco et al., 2007). This effect was also seen here for respectively, which might be evident in side-by-side comparisons of SUMOylation profiles. As shown in Figure S2, the molecular mass distributions of both the smear of conjugates and individual species accumulating both before and after a 30-min heat shock at 37 C were indistinguishable between Col-0 seedlings and the sumo1-1 sumo2-1 seedlings rescued with either WT SUMO1 or K0 transgenes. Taken together, we suggest that such secondary modifications are not prevalent in Arabidopsis even though they can be detected by MS/MS (Miller et al., 2010(Miller et al., , 2013 Yoo et al., 2006). As shown in Figure 3, 7-day-old seedlings of both the K0 and the K23,42-R lines had normal tolerances to such stress.
Although the growth of ami-S2 sumo1-1 seedlings was substantially arrested when acclimated for 7 days at 21 C and then exposed to 35 C for as little as 1 day before returning back to 21 C, all the rescued K0 and K23,42-R seedlings showed strong heat resistance like Col-0 seedlings or sumo1-1 sumo2-1 seedlings rescued with WT SUMO1. In fact, they remained moderately viable even after a 5-day exposure to 35 C ( Figure 3).
We next tested the sensitivity of the mutants to an assortment of stress-inducing chemicals, some of which had been previously connected to the SUMO system (Benlloch & Lois, 2018;Castro et al., 2012;Morrell & Sadanandom, 2019;Xu et al., 2013;Zhang et al., 2021). The list, used at concentrations known to compromise Arabidopsis growth when added to solid medium, included NaCl (100 mM) for salt stress, mannitol (200 mM) for osmotic stress, the DNA-damaging agents bleocin (10 nM) and methyl methanesulfonate (MMS; 5 ppm) that induce genotoxic stress, paraquat (2 nM) that generates cytotoxic reactive oxygen species by blocking photosynthetic electron transport, and the proteasome inhibitor MG132 (20 μM).
Here, subtle but reproducible effects on seedling growth were evident for some effectors after a 7-day treatment when comparing the K0 and K23,42-R lines to Col-0 and seedlings expressing WT SUMO1 ( Figure 4a). Although the lysine-less mutants grew more poorly on NaCl, they consistently grew slightly better on mannitol. Similarly, the mutants were hypersensitive to bleocin, which induces DNA strain scission, but hyposensitive to MMS, which induces DNA damage via deoxyguanosine/deoxyadenosine methylation and stalling at DNA replication forks. The DNA-damaging agents hydroxyurea and mitomycin C were also tested but showed no differential impact on seedling growth when comparing the K0 and K23,24-R mutants to Col-0 even at concentrations (1 to 5 mM and 10 μg/ml, respectively) that substantially impacted seedling growth for Col-0 ( Figure S3A). The lysine-less mutants were also less sensitive to paraquat but surprisingly only marginally impacted by MG132 despite connections of the Ub-proteasome system to SUMO through StUbLs and the assembly of SUMO-Ub polymers (Benlloch & Lois, 2018;Elrouby, 2015;Miller et al., 2010).
As the SUMO system has been intimately linked to plant hormone signaling Benlloch & Lois, 2018;Castro et al., 2012), we also tested the response of the K0 and K23,42-R lines to auxin, salicylic acid (SA), abscisic acid (ABA), and gibberellic acid (GA) and its biosynthesis inhibitor paclobutrazol (PAC), all of which are influenced by SUMOylation (Conti et al., 2014;Ishida et al., 2009;Lois et al., 2003;Miura et al., 2009Miura et al., , 2010Orosa-Puente et al., 2018). Auxin and SA treatments failed to show observable differences between Col-0 and the K0 and K23,42-R lines using primary root elongation as the assay ( Figure S3B). (c) Effect of heat shock on the SUMO conjugate profile in wild-type Col-0 seedlings, and independent transgenic sumo1-1 sumo2-1 lines expressing either wild-type SUMO1 (WT), or the SUMO1(K0) and SUMO1 K23,42-R variants. Seven-day-old seedlings grown under continuous light in liquid medium at 24 C, were subjected at t = 0 to a 30-min heat shock at 37 C (red arrows), followed by incubation at 24 C for additional times. Free SUMO and SUMO conjugates, as detected in total seedling lysates by SDS-PAGE and immunoblotting with anti-SUMO1 antibodies, are located by the arrowhead and bracket, respectively. Immunodetection of histone H3 was used to verify near equal protein loading.
Strikingly, the K0 variants were strongly hypersensitive to ABA while the K23,K42-R lines were modestly hypersensitive, using seed germination, cotyledon greening, and seedling root growth as welldescribed ABA responses, which was consistent with the known connections between ABA signaling and the SIZ1 SUMO E3 (Lois et al., 2003;Miura et al., 2009). While the K0-lines germinated similarly to WT SUMO1 on control medium, they were substantially GA signaling has been linked to the SUMO system through modification of the growth repressing DELLA proteins, which are key hubs within the GA response pathway (Conti et al., 2014;Kim et al., 2015).
Whereas GA sensitivity was unaltered in the K0 and K23,42-R lines as compared with Col-0 and the WT SUMO1 lines based on the percentage of seed germination after 7 days, it was effectively suppressed by PAC (Figure 4b). At 5 μM PAC, germination was strongly or completely suppressed in the K23,42-R line and most of the K0 lines, while 58% and 17% of the Col-0 and WT seeds still germinated, respectively. This suppression could be partially restored by treating the seeds with 10 μM of the bioactive GA isoform GA 3 simultaneous with 5 μM PAC, thus confirming that the dampened seed germination was mediated by inhibited GA synthesis (Figure 4b).
Taken together, it is clear that Arabidopsis can survive and reproduce despite a block in secondary SUMO1/2 modifications. However, we found that the lysine ! arginine replacements did induce more subtle effects on plant growth and development when treated with various agents known to be influenced by SUMOylation.

| Possible use of lysine-null SUMOs to better describe SUMOylation
One complication in attempts to identify SUMOylated proteins and map individual SUMOylation sites by MS/MS-based approaches is the potential complexity of the modified proteins that can bear multiple SUMOs individually attached to varying numbers of lysines within each target, each of which can be modified further with additional F I G U R E 3 Arabidopsis expressing SUMO1(K0) have normal thermotolerance. (a) Treatment regimen testing the thermotolerance to moderately high temperature. Seedlings were grown under a LD photoperiod for 7 days at 21 C on solid GM medium containing 2% sucrose, incubated for 1-6 days at 35 C, and then returned to 21 C for 20 days of total growth before assay of tolerance by continued growth.
(b) Representative plates of seedlings either not exposed to the heat stress (t = 0) or exposed to 35 C for 3 or 5 days before assay. Plates were photographed after 20 days of total growth. Shown are wild-type Col-0 seedlings, and homozygous sumo1-1 sumo2-1 lines expressing under the SUMO1 promoter either wild-type SUMO1 (WT #101), the K23,42-R variant, or three independent lines expressing SUMO1(K0) (K0 #2, K0 #109, and K0 #116). Survival of the amiS2 sumo1-1 line was included for comparison. Scale bar = 1 cm. (c) Quantification of the thermotolerance assay as shown in panel (b). Plotted are the relative growth of seedlings exposed to increasing numbers of days at 35 C before return to 21 C. Each point represents the normalized mean fresh weight from three biological replicates (±SD). Asterisks identify significant differences as compared with Col-0 by two-way ANOVA (p value <.05).
F I G U R E 4 Legend on next page.
SUMOs and/or Ubs (Hendriks et al., 2015;Miller et al., 2010;Vertegaal et al., 2006;Wohlschlegel et al., 2006). Clearly, one way to simplify mapping of SUMOylation sites would be to employ the aforementioned SUMO1 variants bearing lysine ! arginine substitution that can block such secondary modifications while also being amenable to affinity purification and MS/MS detection of SUMO binding sites (Miller et al., 2010;Vertegaal et al., 2006;Wohlschlegel et al., 2006). As shown by Miller et al. (2010Miller et al. ( , 2013, the latter is possi- to 24 C ( Figure 6). By in large, the size distribution and kinetics of F I G U R E 4 Arabidopsis expressing SUMO1(K0) is differentially sensitive to various stress conditions, abscisic acid (ABA), and the gibberellic acid (GA) biosynthetic inhibitor paclobutrazol (PAC). Tested were wild-type Col-0 and homozygous sumo1-1 sumo2-1 seeds/seedlings expressing under the SUMO1 promoter either wild-type SUMO1 (WT #101), the K23,42-R variant, or three independent transgenic lines expressing SUMO1(K0) (K0 #2, K0 #109, and K0 #116). Survival of the amiS2 sumo1-1 line was included for comparison. The dashed lines highlight the mean response of Col-0 to each treatment condition. All percentage values were normalized to those of control seedlings without the treatment. 0 indicates treatments where no seeds germinated (panel b) or where no seedlings greened (panel c). Asterisks in panels (a, b, c, and e) identify significant differences as compared with Col-0 by two-way ANOVA (p value <.05). (a) Sensitivity of seedling growth to either 100 mM NaCl, 200 mM mannitol, 10 nM bleocin, 5 ppm methyl methanesulfonate (mMS), 2 nM paraquat, or 20 μM MG132. Seedlings were grown at 21 C on solid 1/2 MS medium for 3.5 days without the chemicals, transferred to solid medium with the indicated concentrations, and then measured for root growth after 7 additional days. Each bar represents normalized mean response (±SE). n = 6 for MG132 and n = 10 to 16 for the other treatments. (b) Sensitivity of seed germination to GA3, and the GA synthesis inhibitor PAC. Seeds were sown on solid 1/2 MS medium containing the indicated concentrations of GA and/or PAC, stratified at 4 C in darkness for 3 days, and then assayed for germination by radical protrusion following a 7-day incubation at 21 C in white light. Each bar represents the normalized mean percentage germination of two biological replicate plates, each containing 15 seeds (±SE). (c) Sensitivity of cotyledon greening to ABA after germination. Seeds were sown on solid 1/2 MS medium containing the indicated concentrations of ABA, stratified at 4 C in darkness for 3 days, and then visually assayed for green cotyledons after 7 days of incubation under a long-day photoperiod at 21 C. Each bar represents the normalized mean percentage of either four biological replicate plates (control, .5 μM, and 1 μM) or two biological replicate plates (1.5 μM), each containing 15 seedlings (±SE). (d) Germination and growth for 7 days of seedlings treated without or with .5-or 1 μM ABA. Fifteen seeds were plated for each genotype. The dashed lines roughly demarcate lanes where the roots of each line grew. Scale bar = 1 cm. (e) Percentage of seed germination for various days treated without or with 1 μM ABA. Each bar represents the normalized mean percentage of four biological replicate plates, each containing 15 seeds (±SE).
conjugate assembly/disassembly for the SUMO1 and K0 variants were also indistinguishable when comparing the WT #101 and K0 #113 lines ( Figure 6). However, we presume that the SUMO1 variant proteins are probably not completely WT as judged by their dampened assembly into conjugates based on their relative levels versus free SUMO1 seen during the heat shock ( Figure 6).
As a prelude to more in depth proteomic analysis of SUMOylated proteins, we then tested whether these engineered SUMO1 variants could be exploited to affinity purify SUMOylated proteins directly from Arabidopsis. As shown in Figure 7, a single Ni-NTA chromatography step was sufficient to enrich for SUMOylated species from heat- second step would be required for more stringent, in-depth MS/MS studies (e.g., Hendriks et al., 2015;Miller et al., 2010Miller et al., , 2013Rytz et al., 2018).  (Miller et al., 2010(Miller et al., , 2013 and by preliminary genetic studies on potential Arabidopsis StUbLs that were presumed to ubiquitylate SUMOs based on sequence homology (Elrouby et al., 2013;Hale et al., 2016). Here, we attempted to directly address this question through complementation studies that combined a mutant eliminating expression of the two related and essential SUMOs in Arabidopsis (SUMO1 and 2) with a SUMO1 variant in which all lysines were replaced with arginines. Prior studies with this SUMO1(K0) protein F I G U R E 6 6His-(M1R)-SUMO1-K0(H89R) faithfully conjugates to Arabidopsis proteins during heat stress. Seven-day-old seedlings, either wild type (Col-0) or harboring the sumo1-1 sumo2-1 mutations together with the 6His-(M1R)-SUMO1(H89R) and 6His-(M1R)-K0(H89R) transgenes were grown under continuous light in liquid MS medium at 24 C, subjected to a 30 min heat shock at 37 C (red arrows), and then returned back to 24 C for additional times. Total protein extracts were subjected to SDS-PAGE and immunoblot analysis with anti-SUMO1 antibodies. Free SUMO1 and SUMO conjugates are located by the arrowhead and brackets, respectively. Immunoblot analysis with anti-histone H3 antibodies was used to verify near equal protein loading.

| DISCUSSION
F I G U R E 7 Use of an engineering variant of SUMO1 to enrich for SUMOylated proteins from Arabidopsis. Col-0 wild-type seedling or sumo1-1 sumo2-1 seedlings expressing the 6His-(M1R)-SUMO1(H89R) variant encoding either wild-type SUMO1 (WT) or the K0 variant were grown for 7 days in liquid culture followed by a 30-min heat shock at 37 C. The seedlings were then homogenized and subjected to Ni-NTA affinity chromatography. Equivalent protein amounts from the crude extract, flow-through (FT) or eluate (EL) fractions were subjected to SDS-PAGE and either stained for protein with Coomassie blue (left panel) or subjected to immunoblot analysis with anti-SUMO1 antibodies (right panel). Free SUMO1 and SUMO conjugates are located by the arrowhead and brackets, respectively.
showed that it is immune to secondary SUMOylation at least in vitro using purified components (Augustine et al., 2016).
Surprisingly, these SUMO1(K0) sumo1-1 sumo2-1 plants were viable and relatively normal phenotypically when grown under optimal conditions, and showed similar thermotolerance to moderately high temperatures and indistinguishable SUMOylation patterns both before and after a strong heat shock of 37 C. Both the rates of conjugate assembly and disassembly were relatively normal, indicating that the E1 ! E2 ! E3 conjugation cascade and the deSUMOylating enzymes in general can tolerate the lysine ! arginine replacements. A similar set of normal responses were also seen for the K23,K42-R replacement affecting the only lysine modification sites detected thus far in planta (Miller et al., 2010). The SDS-PAGE profiles of K0-and K23,42-R-derived conjugates also appeared unaltered as compared with those of endogenous SUMOs or ectopically expressed WT SUMO1, suggesting either that few Arabidopsis proteins undergo secondary SUMO-SUMO or SUMO-Ub modifications at these lysines or that their apparent molecular masses are insufficiently impacted by the additional SUMOs or Ubs to be seen electrophoretically. Consequently, we are left to propose that secondary modifications of SUMO are relatively minor even if they do occur in planta, as compared with direct modification of substrates through isopeptide addition at one or more substrate lysines. In fact, it is possible that SUMOylation of SUMO1/2 seen previously with Arabidopsis (Miller et al., 2010(Miller et al., , 2013 represents an overzealous conjugation machinery analogous to that proposed for the SUMO 'spray' modification of proteins near the directly intended target (Psakhye & Jentsch, 2012).
The one caveat to our conclusions is the possibility that the noncanonical SUMO isoforms (SUMO3 and 5) could replace the lysine-less SUMO1 functionally as well as in forming SUMO-SUMO and SUMO-Ub secondary modifications, but this seems unlikely given that neither endogenous loci can rescue sumo1-1 sumo1-2 backgrounds (Saracco et al., 2007), while the SUMO1(K0) mutant provides strong replacement (this report). Another scenario, also unlikely, is that other residues beside lysine can provide secondary attachment sites in plants. While no data yet exist for SUMO, accumulating evidence with ubiquitylation in mammalian cells have discovered that other amino acids can be linkage sites. The best understood is the N-terminal methionine amino group, which for Ub has been found to be a peptide-bond acceptor site does elicit a variety of more subtle growth defects when exposed to various stress conditions and hormones, some of which have been linked to SUMOylation through prior genetic analyses of mutants within the SUMO system (Benlloch & Lois, 2018;Castro et al., 2012;Conti et al., 2014;Kim et al., 2015;Lois et al., 2003;Miura et al., 2009;Morrell & Sadanandom, 2019;Xu et al., 2013;Zhang et al., 2021).
Included are a hypersensitivity to DNA damaging agents, a differential sensitivity to salt and maybe osmotic stress, and a hypersensitivity to T A B L E 1 Proteins identified by tandem mass spectrometry with 6His-(M1R)-SUMO(H89R) harboring WT and K0 SUMO1 a .  ABA and the GA-biosynthesis inhibitor PAC. Although it is possible that these effects were caused by a block in specific secondary SUMO modifications that have restricted phenotypic consequences, it is also possible that the arginine replacements selectively impact only a few SUMOylation/deSUMOylation events by directly altering interactions with downstream partners (e.g., proteins with SIMs) either before or after attachment. Whatever their roles, the lysine ! arginine replacements appear relatively inconsequential to Arabidopsis embryo development and seedling growth.  (Miller et al., 2010(Miller et al., , 2013 Preliminary studies showed that these 6His-(M1R)-SUMO1 (H89R) and 6His-(M1R)-K0(H89R) proteins not only enabled enrichment of SUMO conjugates by Ni-NTA chromatography but also allow their detection by MS/MS analysis of the samples. As seen previously Miller et al., 2010Miller et al., , 2013Rytz et al., 2018), many in the catalog of conjugates have direct links to various nuclear functions, including transcription, epigenetic modifications, chromatin structure, and RNA metabolism. However, we acknowledge that additional enrichment steps, possibly using anti-SUMO affinity chromatography (Miller et al., 2010) or the LysC-based PRISM strategy (Hendriks et al., 2015;Miller et al., 2010), will likely be required to generate samples suitable for detailed proteomic analysis of SUMOylation and the mapping to SUMO1/2 attachment sites.
Clearly, these engineered lines should provide valuable new tools for continued investigations of the plant SUMOylome.

| Plant materials and growth conditions
The Arabidopsis thaliana ecotype Columbia (Col-0) was used as the genetic background for all plant lines. The amiS2 sumo1-1 line was as described (van den Burg et al., 2010). Prior to phenotypic studies, all the mutant and transgenic lines were grown simultaneously and the resulting seeds were allowed to after ripen for 2 months at 21 C to establish uniform seed populations with high germination rates.
Unless otherwise noted in the figure legends, seeds were surface sterilized using the vapor-phase method and stratified in water at 4 C for 2 days in the dark before germination on MS medium ( constructions into the pMDC99 plant transformation vector (Curtis & Grossniklaus, 2003), which was then inserted by the floral-dip method into plants heterozygous for sumo1-1 (SAIL_296_C12) and homozygous for sumo2-1 (Salk_129775) (Saracco et al., 2007). T1 seedlings harboring the SUMO1 and K0 transgenes were identified by hygromycin resistance and confirmed by genomic PCR using the EconoTaq Plus Green 2X MasterMix (Lucigen). Selected transformants were allowed to self-cross and T2 seedlings were screened for the SUMO1 transgenes and for homozygosity of the sumo1-1 and sumo2-1 alleles by hygromycin resistance followed by genomic PCR using primers located in Figure 1a. Primers P1 and P2 were used to detect the 2.3-kb transgene.
Primers P3, P4 and P5 were used to detect the sumo1-1 allele, and the P6, P7, and P8 primers were used to detect the sumo2-1 allele.
To construct the 6His-(M1R)-SUMO1(H89R) and 6His-(M1R)-K0 (H89R) lines, the SUMO1 promoter and SUMO1 coding region plus 3'-UTR were amplified separately from the SUMO1 genomic fragment described above with primers designed to add an XbaI restriction site to the 3 0 end of the promoter and an XbaI site followed by the codons that replaced the initiator methionine codon with the MHHHHHHR protein sequence (6His-[M1R]). The 6His-SUMO1 construction was inserted into the XbaI site of the pDNR221-SUMO1 vector, which was then modified to harbor the H89R codon variant as described (Miller et al., 2010). Codons for the N-terminal methionine and the six remaining internal lysines were converted to those for arginines by QuikChange as above. These constructions were introduced into the pMDC100 plant transformation vector by Gateway LR recombination reaction (Curtis & Grossniklaus, 2003) and then inserted into heterozygous sumo1-1, homozygous sumo2-1 plants as described above. T1 seedlings harboring the 6His-(M1R)-SUMO1(H89R) and 6His-(M1R)-K0 (H89R) transgenes were identified by kanamycin resistance followed by genomic PCR and allowed to self-cross to generate T2 plants homozygous for the sumo1-1, sumo2-1, and transgene loci as determined by genomic PCR.

| qRT-PCR analyses
Total RNA was extracted from 7-day-old seedlings using the RNeasy transcript was normalized to that generated with ACT2 based on the comparative threshold method (Pfaffl, 2001).

| Immunoblot analyses and antigenicity test
For SUMO1/2 immunodetection, seedlings were grown in liquid 1/2 MS medium for 7 days at 24 C, heat-shocked at 37 C for 30 min, and then returned for 24 C for various times. Tissue was blotted dry, flash frozen in liquid nitrogen, pulverized by a mortar and pestle, and mixed with two volumes (μl) per milligrams of fresh weight of twice-strength Tween-20, and probed with rabbit anti-Arabidopsis SUMO1 antibodies (Abcam, Cat. No. ab5316) as described (Kurepa et al., 2003). Rabbit antibodies against the histone-3 (Abcam, Cat. No. ab1791) were used to confirm equal loading. The membranes were probed with horseradish peroxidase-decorated goat anti-rabbit secondary antibodies (SeraCare, Cat. No. 5220-0341) used at a dilution of 1:10,000, followed by chemiluminescence signal detection with the SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific) together with X-ray film.
For the antigenicity tests of the anti-SUMO1 antibodies, fragments encoding recombinant WT 6His-SUMO1 and 6His-SUMO1 (K0) in their active forms (i.e., ending in the di-Gly motif) were inserted into the pDEST17 plasmid (Thermo Fisher Scientific) and expressed in Escherichia coli Rosetta (DE3)pLysS cells (EMD Millipore) by overnight induction with isopropyl ß-D-1-thiogalactopyranoside (Sigma), followed by purification using Ni-NTA beads (QIAGEN). Antigenicity was quantified by immunoblot analysis of serial dilutions of the purified proteins with anti-SUMO1 antibodies followed by detection with IRDye 800CW goat anti-rabbit antibodies (LI-COR). Signals were quantified with a LICOR Odyssey Classic FluorImager set at the 800-nm channel, which were normalized against background by the LICOR imaging software.

| Affinity purification of SUMO conjugates
Seven-day-old seedlings cultured in the above-mentioned liquid MS medium were heat-shocked at 37 C for 30 min, blotted dry, and immediately frozen in liquid nitrogen. Approximately 25 g of frozen tissue was pulverized and resuspended at 55 C in extraction buffer (EXB; 100 mM Na 2 HPO 4 , 10 mM Tris-HCl, 300 mM NaCl, and 10 mM iodoacetamide [IAA]) and made 7 M guanidine-HCl, 10 mM sodium metabisulfate, and 2 mM PMSF just before use (final pH 8.0) . The extract was filtered through two layers of Miracloth (EMD Millipore), clarified by centrifugation at 15,000 g, and incubated overnight at 4 C with Ni-NTA resin (Qiagen) (.75-ml resin/5 g of tissue) after addition of imidazole to 10 mM. The Ni-NTA beads were washed sequentially with 10 column volumes of EXB containing 6 M guanidine-HCl, .25% Triton X-100, and 10 mM imidazole (pH 8.0); 10 column volumes of EXB containing 8M urea, .25% TritonX-100, and 10 mM imidazole (pH 6.8); and 15 column volumes of EXB containing 8 M urea, .25% Triton X-100, and 10 mM imidazole (pH 8.0). SUMO conjugates were released with five column volumes of elution buffer containing 350 mM imidazole, 100 mM Na 2 HPO 4 , 10 mM Tris-HCl, and 10 mM IAA (pH 8.0). The eluant was concentrated by ultrafiltration with a 10-kDa molecular mass cutoff filter (Vivaspin; GE Healthcare Life Sciences).

| MS/MS analysis of SUMO conjugates
The Ni-NTA-eluted fractions were processed for MS/MS analysis as described (Rytz et al., 2018). Samples were reduced and alkylation with 20 mM IAA with the reaction quenched with 20 mM dithiotreitol, and then diluted with 25 mM ammonium bicarbonate. The samples were digested overnight at 37 C with .5 μg of sequencing-grade modified porcine trypsin (Promega), lyophilized to a final volume of $250 μl, acidified with .5% (v/v) trifluoroacetic acid (pH < 3.0), and desalted and concentrated using a 100 μl Pierce C18 pipette tip (Thermo Fisher Scientific). Bound peptides were eluted in 50 μl of 75% acetonitrile and .1% acetic acid, lyophilized, and resuspended in 20 μl 5% acetonitrile and .1% formic acid.
The peptides were analyzed with a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) after reversed-phase nanohigh-performance liquid chromatography separation over 135 min with a 25 cm analytical C18 resin column (Acclaim PepMap RSLC; Thermo Fisher Scientific) and a 5%-95% acetonitrile gradient in .1% formic acid at a flow rate of 250 nl/min (Rytz et al., 2018). Peptides were assigned by SEQUEST HT (Eng et al., 1994), allowing a maximum of 2 missed tryptic cleavages, a minimum peptide length of 6, a precursor mass tolerance of 10 ppm, and fragment mass tolerances